The present invention relates to the separation of biomolecules from plasma, particularly human plasma.
Human plasma contains approximately 3000 proteins with a variety of functions and potential therapeutic uses. Tight control of plasma available for blood fractionation means that the supply of important therapeutic agents like IgG is severly curtailed. This together with methodology which ends in very low yields and takes three to five days contributes to the international shortfall of major plasma fractions.
The present inventors have found that rapid isolation times, high recoveries and high-resolution make Gradiflow(trademark) technology a viable alternative purification technology to conventional Cohn precipitation and column chromatography [1, 2].
Albumin and IgG both have enormous importance in medicine and therefore are of considerable commercial value. Albumin alone has an estimated annual global market value of $US1.5 billion [3]. Conventional purification protocols are cumbersome and expensive with low yields and long processing times [4].
Albumin is the most abundant protein component (50 mg/mL) in human plasma and functions to maintain whole blood volume and oncotic pressure. Albumin also regulates the transport of protein, fatty acids, hormones and drugs [4]. Clinical uses include blood volume replacement during surgery, treatment of shock, serious bums and other medical emergencies and the stabilisation of other pharmaceutical products.
Albumin has a molecular mass of 67 kDa and an isoelectric point (pI) of approximately 4.9. The protein consists of a single subunit and is globular in shape [5]. Conventional purification schemes use the Cohn ethanol precipitation method and result in only 50% recovery.
Immunoglobulin G (IgG) is the most abundant of the immunoglobulins, representing almost 70% of the total immunoglobulin component in human serum. The concentration of IgG in normal plasma is approximately 10 mg/mL [6]. The IgG plays an essential role in the immune response and have clinical uses including treatment of snake and spider bites, neurological disorders and IgG is commonly used in analytical or diagnostic kits.
The gamma-globulins have a molecular mass of approximately 150 kDa and consist of four chains, two of which are light and two of which are heavy [6]. Immunoglobulins are traditionally isolated using Cohn ethanol precipitation or alternatively affinity chromatography [7].
Alpha-1-antitrypsin is an acid glycoprotein of 54 kDa with an isoelectric point of 4.8 and is used in the treatment of hereditary emphysema [8]. Conventional purification schemes utilise a combination of Cohn fractionation and column chromatography with the major difficulty being the removal of albumin from xcex1-1-antitrypsin preparations [9]. Current production schemes provide a yield of approximately 30% and much of this is contaminated with albumin. The present inventors have adapted Gradiflow(trademark) to provide an alternative technique for producing highly pure xcex1-1-antitrypsin with a yield of above 70%. This strategy also exemplifies Gradiflow(trademark) technology""s use in isolating protease inhibitors.
Gradiflow(trademark) Technology
Gradiflow(trademark) technology utilises molecular characteristics of size and charge to isolate protein [1] with the resolution of two-dimensional electrophoresis and the throughput of preparative chromatography. Proteins exist as charged molecules above or below their isoelectric point (pI). In the Gradiflow(trademark) the net charge on a macromolecule is controlled by the choice of buffer pH. The proteins are separated in an electric field by charge and/or size differences [2].
The present inventors have found that the Gradiflow(trademark) technology can be adapted to purify a number of different biomolecular components from plasma. The present inventors have devised methodology for the rapid isolation of albumin, IgG and xcex1-1-antitrypsin from a single volume of plasma in a four-phase process with high yield and low cost.
In a general aspect, the present invention relates to the sequential separation of a number of biomolecules present in a plasma sample using four major separation phases or processes.
In a first aspect, the present invention consists in a method of separating components from plasma, the method comprising the steps:
Phase Ixe2x80x94Removal of albumin, xcex1-1-antitrypsin and small contaminants
(a) placing the plasma in a first solvent stream, the first solvent stream being separated from a second solvent stream by a first electrophoretic separation membrane having a molecular mass cut-off less than the molecular mass of albumin and a restriction membrane having a molecular mass cut-off less than the first electrophoretic separation membrane;
(b) selecting a buffer for the first solvent stream having a pH greater than the pI of albumin;
(c) applying an electric potential between the two solvent streams causing movement of albumin and xcex1-1-antitrypsin through the first electrophoretic membrane into the second solvent stream while biomolecules having a molecular mass greater than albumin and xcex1-1-antitrypsin are substantially retained in the first solvent stream, or if entering the first electrophoresis membrane, being substantially prevented from passing through the first electrophoresis membrane, wherein biomolecules in the plasma having a molecular mass less than albumin and xcex1-1-antitrypsin are caused to move through the first separation membrane and the restriction membranes to a waste collection;
(d) optionally, periodically stopping and reversing the electric potential to cause movement of biomolecules having a molecular mass greater than albumin and xcex1-1-antitrypsin having entered the first electrophoresis membrane to move back into the first solvent stream, wherein substantially not causing any albumin or xcex1-1-antitrypsin that have entered the second solvent stream to re-enter first solvent stream;
(e) maintaining steps (c) and optionally (d) until the desired amount of albumin and xcex1-1-antitrypsin have been collected as an albumin/xcex1-1-antitrypsin pool and biomolecules having a molecular mass less than albumin and xcex1-1-antitrypsin have been removed from the first solvent stream to form a treated plasma;
Phase IIxe2x80x94Removal of large contaminants
(f) placing the treated plasma in a third solvent stream, the third solvent stream being separated from a fourth solvent stream by a second electrophoretic separation membrane having a molecular mass cut-off less than the molecular mass of immunoglobulins;
(g) selecting a buffer for the third solvent stream having a pH above neutral;
(h) applying an electric potential between the third and fourth solvent streams causing movement of biomolecules having a molecular mass less that that of immunoglobulins in the treated plasma through the second electrophoretic separation membrane into the fourth solvent stream while immunoglobulins and other biomolecules having a molecular mass greater than immunoglobulins are substantially retained in the third solvent stream, or if entering the second electrophoresis separation membrane, being substantially prevented from passing through the second electrophoresis separation membrane;
(i) optionally, periodically stopping and reversing the electric potential to cause movement of immunoglobulins and other biomolecules having a molecular mass greater than immunoglobulins having entered the second electrophoresis separation membrane to move back into the third solvent stream, wherein substantially not causing any biomolecules having a molecular mass less than immunoglobulins that have entered the fourth solvent stream to re-enter third solvent stream;
(j) maintaining steps (h) and optional (i) until the desired amount of biomolecules having a molecular mass less than immunoglobulins have been removed from the third upstream to form an immunoglobulins concentrate;
(k) removing the biomolecules from the fourth solvent stream;
Phase IIIxe2x80x94separation of immunoglobulins
(l) replacing the second electrophoretic separation membrane with a third electrophoretic separation membrane having a molecular mass cut-off greater than the molecular mass of immunoglobulins;
(m) selecting a buffer for the immunoglobulins concentrate having a pH below neutral;
(n) applying an electric potential between the immunoglobulins concentrate in the third solvent stream and a fresh fourth solvent stream causing movement of immunoglobulins in the immunoglobulins concentrate in the third solvent stream through the third electrophoretic separation membrane into the fresh fourth solvent stream while biomolecules having a molecular mass greater than immunoglobulins are substantially retained in the third solvent stream, or if entering the third electrophoresis separation membrane, being substantially prevented from passing through the third electrophoresis separation membrane;
(o) optionally, periodically stopping and reversing the electric potential to cause movement of biomolecules having a molecular mass greater than immunoglobulins having entered the third electrophoresis membrane to move back into the treated third solvent stream, wherein substantially not causing any immunoglobulins that has entered the fresh fourth solvent stream to re-enter treated third solvent stream;
(p) maintaining steps (n) and optional (o) until the desired amount of immunoglobulins have been moved to the fresh fourth downstream;
Phase IVxe2x80x94Separation of albumin from xcex1-1-antitrypsin
(q) placing the albumin/xcex1-1-antitrypsin concentrate in a fifth solvent stream, the fifth solvent stream being separated from a sixth solvent stream by a fourth electrophoretic separation membrane having a molecular mass cut-off less than the molecular mass of albumin;
(r) selecting a buffer for the fifth solvent stream having a pH greater than neutral;
(s) applying an electric potential between the fifth and sixth solvent streams causing movement of xcex1-1-antitrypsin through the fourth electrophoresis separation membrane into the sixth solvent stream while albumin is substantially retained in the fifth solvent stream, or if entering the fourth electrophoresis separation membrane, being substantially prevented from passing through the fourth electrophoresis separation membrane;
(t) optionally, periodically stopping and reversing the electric potential to cause movement of albumin having entered the fourth electrophoresis separation membrane to move back into the fifth solvent stream, wherein substantially not causing any xcex1-1-antitrypsin that has entered the sixth solvent stream to re-enter the fifth solvent stream; and
(u) maintaining steps (s) and optionally (t) until the desired amount of albumin remains in the fifth solvent stream and the desired amount of xcex1-1-antitrypsin has have been removed to the sixth solvent stream.
As the present invention is directed to the sequential separation of a number of components from plasma, the steps (q) to (u) can be carried out before steps (f) to (p). Initial steps (a) to (e) produces two products, namely albumin/xcex1-1-antitrypsin pool in the downstream and treated plasma in the upstream. Each of these two products are processed further to produce isolated immunoglobulins, albumin and xcex1-1-antitrypsin.
Preferably, albumin, immunoglobulins and xcex1-1-antitrypsin are separated from a pooled human plasma sample.
The present invention is particularly suited for the separation of immunoglobulin G (IgG).
Preferably, the first electrophoresis separation membrane of step (a) has molecular mass cut-off of about 75 kDa and the restriction membrane has a molecular mass cut off of about 50 kDa. Additional membranes may be positioned before, between or after the separation and restriction membranes to further enhance the separation method.
Preferably, the buffer in step (b) has a pH of about 9. A Tris-borate buffer has been found to be particularly suitable for this separation. It will be appreciated, however, that other buffers having a suitable pH range would also be suitable.
Preferably the second electrophoresis separation membrane of step (f) has a molecular mass cut-off of about 200 kDa. The third electrophoresis separation membrane of step (1) preferably has a molecular mass cut-off of about 500 kDa.
Preferably, the buffer of the third solvent stream in step (g) has a pH of about 9 and the buffer of the treated third solvent stream of step (m) has a pH of less than about 5, more preferably about pH 4.6.
Preferably, the fourth electrophoresis separation membrane of step (q) has molecular mass cut-off of about 50 kDa.
Preferably, the buffer in step (r) has a pH of about 8.0. A Tris-borate buffer has been found to be particularly suitable for this separation. It will be appreciated, however, that other buffers having a suitable pH range would also be suitable.
A potential of 250 volts has been found to be suitable for the separation process. Other voltages, higher or lower, would also be suitable for the present invention depending on the separation membrane(s) used, volume of plasma or treated materials to be processed and the speed of separation required.
Preferably, the first and second solvent streams form part of a first Gradiflow(trademark) apparatus and the third and fourth solvent streams form part of a second Gradiflow(trademark) apparatus.
The purified albumin may be concentrated using a Gradiflow(trademark) system incorporating an electrophoresis separation membrane having a molecular mass cut-off less than the molecular mass of albumin in a pH of greater than 8, preferably about pH 8.4.
The benefits of the method according to the first aspect of the present invention are the possibility of scale-up without adversely altering the properties of the plasma components being separated.
The method according to the present invention results in yields of albumin, immunoglobulins, preferably IgG, and xcex1-1-antitrypsin from plasma of at least 70% with a purity of at least 90% from pooled samples of plasma.
The method according to the present invention results in substantially purified or isolated albumin, immunoglobulins, preferably IgG, and xcex1-1-antitrypsin from plasma in less than 1 day, preferably in less than 12 hours, and more preferably in less than 6 hours. The speed of separation and purity of the final components (albumin, immunoglobulins, preferably IgG, and xcex1-1-antitrypsin) provides a great advance over the prior art methods. Not only does the method allow the processing of one sample of plasma to obtain three major components (albumin, immunoglobulins, preferably IgG, and xcex1-1-antitrypsin), the method is fast and extremely efficient.
In a second aspect, the present invention consists in use of Gladiflow(trademark) in the purification and/or separation of albumin, immunoglobulins, preferably IgG, and xcex1-1-antitrypsin from plasma.
In a third aspect, the present invention consists in albumin, immunoglobulins, preferably IgG, and xcex1-1-antitrypsin purified by the method according to the first aspect of the present invention.
In a fourth aspect, the present invention consists in use of albumin, immunoglobulins, preferably IgG, and xcex1-1-antitrypsin according to the third aspect of the present invention in medical and veterinary applications.
The purification of individual components of plasma is an important illustration of the power of Gradiflow(trademark) in isolating products from complex biological solutions.
Throughout this specification, unless the context requires otherwise, the word xe2x80x9ccomprisexe2x80x9d, or variations such as xe2x80x9ccomprisesxe2x80x9d or xe2x80x9ccomprisingxe2x80x9d, will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.
In order that the present invention may be more clearly understood preferred forms will be described with reference to the following drawings.